Polymerase enzymes, such as DNA polymerase, RNA polymerase, or reverse transcriptase, can catalyse the formation of polynucleotides of DNA or RNA using an existing strand of DNA or RNA as a template. RNA polymerases and DNA polymerases can catalyse the polymerisation of RNA and DNA respectively using a DNA template, whereas reverse transcriptase can catalyse the formation of DNA using an RNA template.
DNA polymerases, for example are naturally occurring intracellular enzymes, and are used by a cell to replicate a nucleic acid strand using a template molecule to manufacture a complementary nucleic acid strand. DNA polymerases are also widely used in vitro for various biochemical applications including cDNA synthesis and DNA sequencing reactions, amplification of nucleic acids by methods such as the polymerase chain reaction (PCR) and for RNA transcription-medicated amplification methods.
The polymerase chain reaction (PCR) is a widely used technique that allows a specific region of DNA to be amplified exponentially, provided that at least part of its nucleotide sequence is already known. This known region of sequence is used to design synthetic DNA oligonucleotides complementary to each strand of the DNA double helix. These oligonucleotides serve as primers for in vitro DNA synthesis, which is catalyzed by DNA polymerase.
Unfortunately, the effectiveness of this technique in basic research or in forensic or clinical applications is limited by some technical problems. A number of substances are known that are potent inhibitors of polymerase activity and limit the use of polymerase chain reaction (PCR) in biological samples where they are present. Examples include heme (and its degradation products such as bilirubin) present in blood and faeces. Another potent inhibitor present in the environment is humic acid.
Humic acids are a complex mixture of polyphenolic acids produced by the decomposition of organic matter (e.g. decomposing terrestrial vegetation). Humic acids are ubiquitous in soil and water and thus are present in any sample exposed to the environment. Inhibition of PCR by humic acids is thus especially relevant for samples of paelontological, archaeological or forensic interest, which are exposed to soil for extended periods of time.
Some attempts have been made to circumvent the problem of humic acid contamination in PCR samples. One approach to the problem has been purification or extraction of DNA from samples in advance of PCR (LaMontagne et al (2002) J Microbiol Methods 49:255-64; Howeler et al J Microbiol Methods 2003 54:37-45). Unfortunately, humic acid contamination may still be a problem depending on the extraction method used (LaMontagne et al 2002). Furthermore, not all contaminants are completely removed during classical extraction protocols (such as detergent, protease and phenol-chloroform treatments), and loss of the original sample may occur. Another problem with extraction procedures includes the use of expensive materials such as ion-exchange columns, glass bead extraction, immunomagnetic separation, size-exclusion chromatography, anion-binding resins or spin columns (Wilson, I G (1997) Appl. Environ. Microbiol. 63:3741-3751). Moreover, the extra steps required in each PCR protocol may increase cross-contamination risks and subsequent false-positive results.
Another approach to tackle the inhibitory effect of humic acid on polymerase activity is to increase the concentration of polymerase in each reaction mixture (Sutlovic et al Croat Med J 2005 46:556-62). Various additives such as BSA, T4 gp32 or salmon sperm DNA are also reported to relieve inhibition of polymerase activity, but these need to be added at substantial concentrations (typically greater than 0.2 mg/ml) (Tebbe et al Appl Environ Microbiol. (1993) 59:2657-65).
With all previous attempts to avoid inhibition of PCR by humic acid—such as extraction techniques or the addition of supplements—extra time and expense is associated with the use of additional reagents or protocols.
There remains a need in the art for a simple and more effective way of dealing with the inhibitory effect of humic acid on DNA polymerases, particularly in PCR.